Recent studies have found that bone marrow-derived endothelial progenitor cells (EPCs) are present in the systemic circulation and home to sites of ischemic injury where they function to promote neovascularization. Cardiovascular diseases characterized by increased reactive oxygen species (ROS) stress and reduced NO bioavailability are associated with reductions in the number and functional activity of circulating EPC, and these reductions may diminish their angiogenic and reendothelialization capacity. EPCs share antigenic markers with hematopoietic stem cells and differentiate into the endothelial lineage in vitro. A number of cytokines, growth factors and adhesion molecules have been implicated in the mobilization of EPCs from the bone marrow stem cell niche and in their homing and differentiation at sites of vascular damage. However, the signaling and transcript!onal regulatory mechanisms that control EPC behavior are incompletely understood. The PI3K/Akt signaling pathway is an important regulator of growth responses in a number of systems, and some evidence has implicated this signaling pathway in promoting the proliferation and differentiation of EPCs under conditions of basal ROS-mediated signaling and compensated oxidant stress. PI3K/Akt signaling contributes to the regulation of the FOXO transcription factors that play a role in cellular resistance to oxidant stress. The proposed studies will test the hypothesis that FOXO transcription factors control EPC proliferation and differentiation and regulate their resistance to oxidant stress. To achieve these objectives, we will first characterize the FOXO isoforms in EPCs with respect to their expression, regulation by ROS and subcellular localization (Aim 1). We will then use gain-of-function and loss-of-function strategies to determine the role of FOXO isoforms in controlling EPC function in vitro including proliferation, differentiation, migration, apoptosis and resistance to oxidant stress (Aim 2). Finally, we will construct lines of transgenic mice that conditionally express constitutivelyactive and dominant-negative FOXO isoforms in EPC and examine the implications for EPC behavior and response to ischemic injury (Aim 3). These studies should provide mechanistic information on the FOXO-mediated regulatory pathways that control EPC phenotype and the relationships between redox stress and EPC function.